阅读说明：本技术 一种艰难梭菌特异性抗原肽 (Clostridium difficile specific antigen peptide ) 是由 赵建宏 李志荣 张慧敏 于 2021-08-04 设计创作，主要内容包括：本发明涉及一种艰难梭菌特异性抗原肽,特别是针对高产毒艰难梭菌RT027的特异性抗原肽,其包含如SEQ ID No.1所示的氨基酸序列。利用本发明的特异性肽段及其特异性抗体,可以开发新的艰难梭菌高产毒株检测试剂盒和艰难梭菌疫苗,对于艰难梭菌高产毒株感染的检测和治疗具有重要意义。(The invention relates to a clostridium difficile specific antigen peptide, in particular to a specific antigen peptide aiming at high-toxicity clostridium difficile RT027, which comprises an amino acid sequence shown as SEQ ID No. 1. By utilizing the specific peptide segment and the specific antibody thereof, a new clostridium difficile high-yield strain detection kit and a clostridium difficile vaccine can be developed, and the kit and the vaccine have important significance for detecting and treating the infection of the clostridium difficile high-yield strain.)

1. Clostridium difficile-specific antigenic peptide, comprising the amino acid sequence shown in SEQ ID No. 1.

2. A nucleic acid molecule encoding a clostridium difficile-specific antigenic peptide of claim 1, comprising the nucleotide sequence shown in SEQ ID No. 2.

3. A vector comprising the nucleic acid molecule of claim 2.

4. An antibody produced using the clostridium difficile-specific antigenic peptide of claim 1.

5. A vaccine prepared using the clostridium difficile-specific antigenic peptide of claim 1 for stimulating an immune response in a host organism.

6. Clostridium difficile specific antigen protein, which is a membrane protein comprising an amino acid sequence shown in SEQ ID No. 1.

7. Clostridium difficile-specific antigenic protein as claimed in claim 6, characterized in that it comprises the amino acid sequence shown in SEQ ID No. 3.

Technical Field

The invention relates to a clostridium difficile specific antigen peptide.

Background

Clostridium difficile (C.difficile) ((C.difficile))Clostridioides difficileCD) is an obligate anaerobe that stains positively with gram-positive spores. CD is a major pathogen of hospital Antibiotic-associated diarrhea (AAD), and can cause mild to severe diarrhea, colitis, pseudomembranous enteritis, toxic megacolon, etc., and even death in severe cases. In recent years, the emergence of high-virulent Clostridium difficile (RT 027/NAP1/BI/ST 01) led toThe morbidity and mortality of Clostridium Difficile Infection (CDI) is increasing, causing tens of thousands of people to die worldwide each year, with treatment costs as high as billions of dollars.

In 2013, the CDC (center for Disease Control and preservation) of Clostridium difficile was the first of the "public health threat by microorganisms" urgency scale. Difficile mainly enters a human body through spores through a feces-oral route and is planted in colon, when the intestinal flora is unbalanced due to long-term use of broad-spectrum antibiotics or proton pump inhibitors and the like, the difficile is propagated in large quantities to generate enterotoxin A (TcdA) and cytotoxin B (TcdB), so that the cell skeleton of the intestinal tract is damaged, the cell permeability is increased, and finally, the disease symptoms such as diarrhea and the like are caused. The pathogenesis of c.difficile infection (CDI) is largely attributed to both toxins. Enhanced systemic humoral immune responses to these toxins have been shown to have a protective effect against recurrent CDI.

Over the years, fully human monoclonal antibodies against both toxins have been developed in an attempt to combat the increasing incidence of recurrent CDI. Vaccination trials have also been studied using two toxins to prepare vaccines. However, current clinical studies using vaccines prepared with toxin a and toxin B show that while 93.3% of volunteers produce more than 2-fold antibodies to toxin a, 82.2% of volunteers produce more than 2-fold antibodies to toxin B. However, there was no significant difference in the incidence of CDI between the vaccinated and control groups, indicating that the vaccine was not significantly effective. Other vaccine trials are in progress, and it is sometimes too early to conclude that their efficacy is also present.

Current research suggests that in addition to two clostridium difficile toxins, their flagella, pili and portions of surface proteins may also play important roles as virulence factors in the colonization and pathogenesis of clostridium difficile. For example, cell wall binding proteins (CWPs) Cwp66, Cwp8, Cwp2, etc. are important adhesion factors involved in colonization; cysteine protease Cwp84 is involved in the cleavage of surface proteins and fibrin; flagellin-related proteins also affect virulence expression. It has been shown that removal of certain surface proteins or treatment of clostridium difficile with corresponding antibodies can reduce colonization by clostridium difficile. Antibodies to surface S layer (SlpA) of clostridium difficile can abrogate clostridium difficile adhesion to mouse 929 and human HeLa cells. After hamster immunization with the cell wall protein Cwp84 as a vaccine, clostridium difficile colonizes intestinal tracts in a hamster infection model immunized with Cwp84 and the overall survival rate is improved compared with an unimmunized control group. However, the results of the current immunological tests on these surface-associated proteins show that these proteins still cannot achieve the effect of effectively clearing clostridium difficile, and it is urgently needed to find more immunogenic target proteins for preparing clostridium difficile vaccines in order to prepare specifically and effectively clostridium difficile vaccines.

Disclosure of Invention

The invention aims to provide a novel clostridium difficile specific antigen peptide with immunogenicity.

The invention adopts the following technical scheme:

a clostridium difficile specific antigen peptide, in particular to a specific antigen peptide aiming at high-toxin clostridium difficile RT027, which comprises an amino acid sequence shown as SEQ ID No. 1.

A nucleic acid molecule encoding the clostridium difficile-specific antigenic peptide as described above, comprising a nucleotide sequence as shown in SEQ ID No. 2.

A vector comprising a nucleic acid molecule as described above.

An antibody prepared by using the clostridium difficile specific antigen peptide.

A vaccine prepared using the clostridium difficile-specific antigenic peptide as described above, for stimulating an immune response in a host organism.

A clostridium difficile specific antigen protein which is a membrane protein comprising an amino acid sequence shown as SEQ ID No. 1.

Further, the clostridium difficile specific antigen protein comprises an amino acid sequence shown as SEQ ID No. 3.

The invention has the beneficial effects that: by utilizing the specific peptide segment and the specific antibody thereof, a new clostridium difficile high-yield strain detection kit and a clostridium difficile vaccine can be developed, and the kit and the vaccine have important significance for detecting and treating the infection of the clostridium difficile high-yield strain.

Drawings

FIG. 1 shows differential protein bands of RT027 and RT017 type C.difficile membrane proteins.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

1. Culture of Clostridium difficile

The invention selects 4 strains of high-yield high-toxicity clostridium difficile RT027 type strains (RT 027/NAP1/BI/ST 01) and 3 strains of low-yield high-toxicity clostridium difficile RT017 type strains for bacterial culture. Before the strain is used, the frozen strain is thawed at room temperature, inoculated in a CDMN selective culture medium, and streaked in a three region, and then cultured in an anaerobic tank at 37 ℃ for 48-72 h. Selecting single colony to be transferred into CDMN culture medium for separation and purification.

2. Extraction of clostridium difficile membrane protein

For the strains that produced membrane proteins, freshly cultured Clostridium difficile cells were washed 3 times with sterile 0.1 mol/L PBS. EZ-Link Sulfo-NHS-SS-biotin reaction solution with the concentration of 250 mu g/ml is prepared and is used for marking the freshly washed bacterial cell membrane protein. The labeled bacterial cells were sonicated (3 s on/7s off, 20% intensity, 5 min). Centrifuging at 12,000 g for 30 min at 4 deg.C, and collecting supernatant rich in difficile mycoprotein. Then, the membrane protein was purified using an avidin protein chromatography column.

3. And (3) performing polyacrylamide gel electrophoresis on the extracted membrane protein, obtaining a corresponding band spectrum of each strain after Coomassie brilliant blue staining, and performing in-gel enzymolysis and mass spectrometry on the difference gel bands of the two strains (see figure 1).

4. Analyzing the difference of protein types and peptide fragments in the difference bands

After the mass spectrum identification of the difference gel bands of the high-yield strain RT027 and the low-yield strain RT017 at the level of 15KD, 225 proteins are obtained. There are 9 specific proteins in the RT027 panel after de-duplication, including transport proteins (CDIF 1296T _02941 etc.), metabolic proteins (atpF etc.), adhesion proteins (fliL, pilin) and other membrane proteins. After the duplication removal, 3 specific proteins including transport proteins (IM 33_ 15590) and the like exist in the RT017 group. Quantitative analysis was carried out based on the primary mass spectrum signal (peak area) of the characteristic peptide fragment, and as a result, it was found that the peptide fragment (SEQ ID No. 1: SASLSYYADESK) of pilin (pilin) was present in RT027 strain in a characteristic manner as a specific differential peptide fragment. The nucleotide sequence corresponding to the difference peptide fragment is SEQ ID No. 2: GCAGATGATTTTACTGCTGATAATTTAAAG are provided.

The protein to which the peptide fragment belongs is pilin (pilin protein: C9YNN 2). The sequence is SEQ ID No. 3: MKNKKGFTLVELLVVIAIIGILAIIALPALFKNIEKAKIAKLEADISAIKSASLSYYADESKYTDGGMISWVKKDGKIIINGGFKDDPLADKIENLGMPYNGSYLLMSSPGHEKYLELSILPEGEISKSGLDKLKNDYGNLIDITNDQNKINIVIKLLNNKSNT

5. Synthesis of a variant peptide according to the identified sequence

Amino acids A-0.527g \ D-0.696g \ E-0.72g \ L-0.598g \ K-0.793g \ S-0.649g \ Y-0.778g are precisely weighed and respectively dissolved in 4ml of DMF overnight. DiEA 56ml was measured carefully and diluted to 200ml with DMF. HBTU-32.1g HOBT-11.4g was precisely weighed, mixed and dissolved in DMF to a volume of 200 ml. Piperidine 60ml was measured precisely and made up to 200ml in DMF. 0.05g of an amino resin was precisely weighed. And starting synthesis after the reagent and the amino acid are completely dissolved.

The following steps are repeated, and corresponding amino acids are added in sequence to synthesize the peptide segment: swelling the amino resin; removing FOMC; eluting; adding amino acid; adding a mixed solvent of HBTU and HOBT; adding DIEA; and (4) eluting. Eluting with DCM again after peptide chain synthesis, performing polypeptide cleavage, eluting with glacial ethyl ether, and lyophilizing the precipitate to obtain corresponding peptide segment.

6. Preparation of antibodies

Balb/c mice were purchased and acclimatized for one week. Weighing, and selecting 5 mice with similar body weights. The synthesized freeze-dried peptide fragment sample is dissolved by pure water, and the carrier protein KLH (10 mg/L) and the polypeptide (4 mg/ml) are mixed uniformly according to the volume of 2: 1 for immunization. The well-mixed polypeptide solution was dialyzed with PBS (pH 7.4) for a minimum of 3 times, with intervals of 3 hours. Finally, the volume is determined to be 3.3ml by filtered PBS, and the mixture is subpackaged into 3 tubes, wherein each tube contains 1.1ml and is stored at the temperature of minus 20 ℃. The immune concentration of the polypeptide is 100 ug/mouse.

When a mouse is immunized, 1 tube is taken, placed and recovered to room temperature, and then mixed with Freund's complete adjuvant in a ratio (polypeptide solution: complete adjuvant = 1: 1.5), after complete emulsification, the back skin of the mouse is grabbed by the left hand, the abdomen is disinfected, the head is fixed at the lower position, the right hand is used for carrying out intraperitoneal injection (falling empty feeling, no liquid is pumped back), 0.5ml is injected into each mouse, and the first immunization is carried out. And in the second and third immunizations, mixing the two with Freund's incomplete adjuvant at a ratio of 1: 1.5. Three total immunizations, 2 weeks between immunization 1 and 2, and 2 weeks between immunization 2 and 3. 7-10 days after 3 rd immunization, the eyeballs are picked and blood is taken, 3500-4000-turn centrifugation is carried out, the supernatant is taken, and the titer is detected by adopting an ElISA method. It was found that antibodies with a titer of greater than 1: 1000 could be obtained after immunological discovery. The peptide fragment is proved to be immunogenic.

SEQUENCE LISTING

<110> second Hospital of Hebei medical university

<120> Clostridium difficile specific antigenic peptide

<130> 2021

<160> 3

<170> PatentIn version 3.3

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<213> Artificial Synthesis

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Ser Ala Ser Leu Ser Tyr Tyr Ala Asp Glu Ser Lys

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<212> DNA

<213> Artificial Synthesis

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gcagatgatt ttactgctga taatttaaag 30

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<212> PRT

<213> Clostridioides difficile

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Met Lys Asn Lys Lys Gly Phe Thr Leu Val Glu Leu Leu Val Val Ile

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Ala Ile Ile Gly Ile Leu Ala Ile Ile Ala Leu Pro Ala Leu Phe Lys

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Asn Ile Glu Lys Ala Lys Ile Ala Lys Leu Glu Ala Asp Ile Ser Ala

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Ile Lys Ser Ala Ser Leu Ser Tyr Tyr Ala Asp Glu Ser Lys Tyr Thr

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Asp Gly Gly Met Ile Ser Trp Val Lys Lys Asp Gly Lys Ile Ile Ile

65 70 75 80

Asn Gly Gly Phe Lys Asp Asp Pro Leu Ala Asp Lys Ile Glu Asn Leu

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Gly Met Pro Tyr Asn Gly Ser Tyr Leu Leu Met Ser Ser Pro Gly His

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Glu Lys Tyr Leu Glu Leu Ser Ile Leu Pro Glu Gly Glu Ile Ser Lys

115 120 125

Ser Gly Leu Asp Lys Leu Lys Asn Asp Tyr Gly Asn Leu Ile Asp Ile

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Thr Asn Asp Gln Asn Lys Ile Asn Ile Val Ile Lys Leu Leu Asn Asn

145 150 155 160

Lys Ser Asn Thr